This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. Abnormal gene expression is a dominant theme in human skin disease and may result from altered levels of normal proteins or expression of defective proteins. Recent experimental breakthroughs in this area, such as targeted gene transcription using keratin promoters in transgenic mice, remain limited by unregulated levels of constitutive gene expression through development. We plan, therefore, to develop the ability to regulate the magnitude of target gene expression, through physiologic and pathologic ranges, with temporal exactness in cutaneous tissue. Such a leap forward in gene regulation will assist the generation of powerful new transgenic models of human skin disease, where primary pathogenic events may be ordered and separated from downstream phenomena, as well as contribute to the development of sustained and regulated cutaneous gene therapy. Precise regulation of the magnitude of target gene expression in intact cells will unleash the quantitative power of conventional biochemistry into the largely qualitative field of genetics. Because methods currently available to attempt this have serious limitations, we will utilize two new advances in our laboratory: (1) synthetic ligand-driven transcription where the otherwise biologically inert reversible hydrophobic cross linker FK1012 can dimerize inactive monomeric proteins into transcription activators, thereby activating target gene expression in a dose-dependent fashion and 2) transcription unit protecting elements, such as BRG1, that may act by preventing unwanted gene inactivation. First, because promoters often exhibit vast differences in activity in different tissue cell types, we will begin by optimizing synthetic ligand-driven transcription separately in primary human keratinocytes and fibroblasts in vitro. A second effort addressing undesired gene inactivation, a problem plaguing many current cutaneous gene delivery efforts, will involve flanking transcription units either with tethering elements for the BRG1 complex or newly defined insulator elements followed by determination of the optimal protecting flanking element in stable transfectants in vitro. Based on information from both approaches, regulated and insulated expression constructs for three epitope tagged prototype genes - arylsulfatase C and dominant-negative K14 for keratinocytes and Factor IX for fibroblast - will be generated, stably transfected into immortalized skin cells and their regulated and sustained expression determined. Finally, we will graft stably transfected keratinocytes and fibroblasts onto SCID mice and assess synthetic ligand-regulated cutaneous and systemic gene delivery. In the case of graded mutant K14 expression in vitro and in intact epidermis, these studies will allow definition of mutant gene dosage necessary to disrupt the intermediate filament network in living keratinocytes and generate a model of regulated human skin disease in vivo. At the end of this five year granting period, we hope to have developed a general approach to achieve regulated and sustainable target gene expression in keratinocytes and fibroblasts with direct application to human cutaneous gene therapy and to the establishment of increasingly powerful transgenic mouse models of human skin disease.